Microfabricated sleeve devices for chemical reactions

ABSTRACT

A silicon-based sleeve type chemical reaction chamber that combines heaters, such as doped polysilicon for heating, and bulk silicon for convection cooling. The reaction chamber combines a critical ratio of silicon and non-silicon based materials to provide the thermal properties desired. For example, the chamber may combine a critical ratio of silicon and silicon nitride to the volume of material to be heated (e.g., a liquid) in order to provide uniform heating, yet low power requirements. The reaction chamber will also allow the introduction of a secondary tube (e.g., plastic) into the reaction sleeve that contains the reaction mixture thereby alleviating any potential materials incompatibility issues. The reaction chamber may be utilized in any chemical reaction system for synthesis or processing of organic, inorganic, or biochemical reactions, such as the polymerase chain reaction (PCR) and/or other DNA reactions, such as the ligase chain reaction, which are examples of a synthetic, thermal-cycling-based reaction. The reaction chamber may also be used in synthesis instruments, particularly those for DNA amplification and synthesis.

RELATED APPLICATION

This application is a continuation-in-part of U.S. application Ser. No.08/492,678, filed Jun. 20, 1995 now Pat. No. 5,589,136 issued Dec. 31,1996.

The United States Government has rights in this invention pursuant toContract No. W-7405-ENG-48 between the United States Department ofEnergy and the University of California for the operation of LawrenceLivermore National Laboratory.

BACKGROUND OF THE INVENTION

The present invention relates to instruments for chemical reactioncontrol and detection of participating reactants and resultant products,particularly to integrated microfabricated instruments for performingmicroscale chemical reactions involving precise control of parameters ofthe reactions, and more particularly to silicon-based and non-siliconbased sleeve devices as reaction chambers for chemical reactions andwhich can be utilized in large arrays of individual chambers for ahigh-throughput microreaction unit.

Current instruments for performing chemical synthesis through thermalcontrol and cycling are generally very large (table-top) andinefficient, and often they work by heating and cooling of a largethermal mass (e.g., an aluminum block). In recent years efforts havebeen directed to miniaturization of these instruments by designing andconstructing reaction chambers out of silicon and silicon-basedmaterials (e.g., silicon, nitride, polycrystalline silicon) that haveintegrated heaters and cooling via convection through the silicon.

Microfabrication technologies are now well known and include sputtering,electrode position, low-pressure vapor deposition, photolithography, andetching. These and similar processes can be applied to the fabricationof reaction chambers and their control elements such as heaters,thermocouples, detectors, sensors, electrodes, and other devices thatcan be used to sense and control the reaction parameters. Examplesinclude magnetic films, thermoelectric films, and electroactive filmsfor reagent manipulation. Additional fabrication techniques includeevaporation, extrusion, casting, sintering, injection, forming, pulling,laminating, etc. can be used to microfabricate reaction chambers out ofa variety of appropriate materials. Microfabricated devices are formed,for example, on crystalline substrates, such as silicon and galliumarsenide, but may be formed on non-crystalline materials, such as glass,ceramic, metals, or certain polymers. The shapes of crystalline silicondevices, for example, can be precisely controlled since etched surfacesare generally crystal planes, and crystalline materials may be bonded byprocesses such as fusion at elevated temperatures, anodic bonding, orfield-assisted methods.

Monolithic microfabrication technology now enables the production ofelectrical, mechanical, electromechanical, optical, chemical and thermaldevices, including pumps, valves, heaters, mixers, and detectors formicroliter to nanoliter quantities of gases, liquids, and solids. Also,optical waveguide probes and ultrasonic flexural-wave sensors can now beproduced on a microscale. The integration of these microfabricateddevices into a single systems allows for the batch production ofmicroscale reactor-based analytical instruments. Such integratedmicroinstruments may be applied to biochemical, inorganic, or organicchemical reactions to perform biomedical and environmental diagnostics,as well as biotechnological processing and detection.

The operation of such integrated microinstruments is easily automated,and since the analysis can be performed in situ, contamination is verylow. Because of the inherently small sizes of such devices, the heatingand cooling can be extremely rapid. These devices have very low powerrequirement and can be powered by batteries or by electromagnetic,capacitive, inductive or optical coupling.

The small volumes and high surface-area to volume ratios ofmicrofabricated reaction instruments provide a high level of control ofthe parameters of a reaction. Heaters may produce temperature cycling orramping; while sonochemical and sonophysical changes in conformationalstructures may be produced by ultrasound transducers; andpolymerizations may be generated by incident optical radiation.

Synthesis reactions, and especially synthesis chain reactions such asthe polymerase chain reaction (PCR), are particularly well-suited formicrofabrication reaction instruments. PCR can selectively amplify asingle molecule of DNA (or RNA) of an organism by a factor of 10⁶ to10⁹. This well-established procedure requires the repetition of heating(denaturing) and cooling (annealing) cycles in the presence of anoriginal DNA target molecule, specific DNA primers, deoxynucleotidetriphosphates, and DNA polymerase enzymes and cofactors. Each cycleproduces a doubling of the target DNA sequence, leading to anexponential accumulation of the target sequence.

The PCR procedure involves: 1) processing of the sample to releasetarget DNA molecules into a crude extract; 2) addition of an aqueoussolution containing enzymes, buffers deoxyribonucleotide triphosphates(dNTPS), and aligonudeotide primers; 3) thermal cycling of the reactionmixture between two or three temperatures (e.g., 90-96, 72, and 37-55°C.); and 4) detection of amplified DNA. Intermediate steps, such aspurification of the reaction products and the incorporation ofsurface-bending primers, for example, may be incorporated in the PCRprocedure.

A problem with standard PCR laboratory techniques is that the PCRreactions may be contaminated or inhibited by the introduction of asingle contaminant molecule of extraneous DNA, such as those fromprevious experiments, or other contaminants, during transfers ofreagents from one vessel to another. Also, PCR reaction volumes used instandard laboratory techniques are typically on the order of 50microliters. A thermal cycle typically consists of four stages: heatinga sample to a first temperature, maintaining the sample at the firsttemperature, cooling the sample to a second lower temperature, andmaintaining the temperature at that lower temperature. Typically, eachof these four stages of a thermal cycle requires about one minute, andthus to complete forty cycles, for example, is about three hours. Thus,due to the large volume typically used in standard laboratoryprocedures, the time involved, as well as the contaminationpossibilities during transfers of reagents from one vessel to another,there is clearly a need for microinstruments capable of carrying out thePCR procedure.

Recently, the cycling time for performing the PCR reaction has beenreduced by performing the PCR reaction in capillary tubes and using aforced air heater to heat the tubes. Also, an integrated microfabricatedreactor has been recently developed for in situ chemical reactions,which is especially advantageous for biochemical reactions which requirehigh-precision thermal cyding, particularly DNA-based manipulations suchas PCR, since the small dimensions of microinstrumentation promote rapidcycling times. This microfabricated reactor is described and claimed incopending U.S. application Ser. No. 07/938,106, filed Aug. 31, 1992 nowU.S. Pat. No. 5,639,423, entitled “Microfabricated Reactor”, now Pat.No. 5,639,423 issued Jun. 17, 1997, assigned to the same assignee. Also,an optically heated and optically interrograted micro-reaction chamber,which can be utilized, for example, in the integrated microfabricatedreactor of the above-referenced copending application Ser. No.07/938,106, now Pat. No. 5,639,423 has been developed for use inchemical reactors, and is described and claimed in copending U.S.application Ser. No. 08/489,819, filed Jun. 13, 1995, entitled DiodeLaser Heated Micro-Reaction Chamber With Sample Detection Means”,assigned to the same assignee.

The present invention is directed to a particular geometry ofsilicon-based and non-silicon-based micro-reactors that have shown to bevery efficient in terms of power and temperature uniformity. Themicro-reactor of this invention, which is broadly considered as asilicon-based or non-silicon-based sleeve device for chemical reactions,can be effectively utilized in either of the reactor systems of theabove-referenced copending applications. The present invention utilizes,for example, doped polysilicon for heating and bulk silicon forconvective cooling. The present invention allows the multi-parameter,simultaneous changing of detection window size, in situ detection,reaction volumes, thermal uniformity, and heating and cooling rates. Inaddition, it enables the use of large arrays of the individual reactionchambers for a high-throughput microreaction unit.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide an improved chemicalreaction chamber.

A further object of the invention is to provide a silicon-based ornon-silicon-based sleeve type chemical reactor.

A further object of the invention is to provide a microfabricatedreactor that uses a combination of materials.

A further object of the invention is to provide a chemical reactionchamber that combines to use of doped polysilicon and bulk silicon.

A further object of the invention is to provide a microfabricatedchemical reactor having a sleeve reaction chamber with a slot thereinfor introducing reaction fluids, either directly or via a tube.

A further object of the invention is to provide silicon or non-siliconreaction sleeves that combines critical ratios of materials to providethe proper thermal characteristics.

A further object of the invention is to provide silicon or non-siliconreaction sleeves that combine a critical ratio of materials to providecontrol of reagents and products.

A further object of the invention is to provide silicon or non-siliconreactions sleeves that combine a critical ratio of materials to providethe proper thermal response.

A further object of the invention is to provide chemical reactionchambers that combines, for example, the use of doped polysilicon andbulk silicon to provide flexibility in thermal and optical propertiesallowing the implementation into small and large instruments.

Another object of the invention is to provide an interconnected seriesor array of silicon or non-silicon reaction sleeves thereby providing aflow-through reaction system.

Another object of the invention is to provide a silicon-based reactionsleeve that combines a critical ratio of silicon and silicon nitride tothe volume of material to be heated (e.g., liquid) in order to provideuniform heating, yet low power requirement.

Another object of the invention is to provide a sleeve reaction chamberthat will allow the introduction of an insert (e.g., plastic) into thereaction sleeve that contains the reaction mixture, thereby elevatingany potential materials incompatibility issues.

Another object of the invention is to provide an interconnected seriesor array of silicon or non-silicon reaction sleeves connected by tubingof polymers, metals, glasses, and ceramics, similar to a “string ofbeads” in appearance.

Another object of the invention is to provide an array of individualreaction chambers for a high-throughput microreaction unit.

Another object of the invention is to provide a hand-held instrumentthat uses sleeve-type reaction chambers with integrated heaters.

Another object of the invention is to provide a reaction chamber withautomated detection and feedback control.

Another object of the invention is to provide for artificialintelligence control of reactions in a reaction chamber.

Another object of the invention is to provide pulse-width modulation asa feedback control for reaction chamber.

Another object of the invention is to provide reaction control withmagnetic films, thermoelectric films, or electroactive films, such aselectrodes.

Another object of the invention is to provide a combination of detectionmodules, such as electrochemiluminescence, optical, electrical andcapacitive.

Another object of the invention is to provide a combined reactionchamber with microelectrophoresis channels.

Another object of the invention is to provide a reaction chamber madefrom silicon and non-silicon directly coupled with microelectrophoresischannel made of silicon or non-silicon.

Another object of the invention is to provide a sleeve-type reactionchamber with microelectrophoresis channel coupled via an internal liner.

Another object of the invention is to provide a microelectrophoresisdetection system based on optical, electrical, or magnetic devices.

Another object of the invention is to provide a microelectrophoresisdetection system based on light emitting diodes and photodiodes.

Other objects and advantages of the present invention will becomeapparent from the following description and the accompanying drawings.Basically, the invention is a silicon-based or non-silicon-based sleevefor chemical reactions. The invention encompasses a chemical reactionchamber that combines, for example, the use of polysilicon for heatingand bulk silicon for convective cooling. The reaction chamber maycombine a critical ratio of non-silicon and silicon based materials toprovide the thermal properties desired. The silicon-based reactionsleeve, for example, may combine a critical ratio of silicon and siliconnitride to the volume of material to be heated in order to provideuniform heating, yet low power requirements. The reaction sleeve of thisinvention also allows for the introduction therein of a secondary tubeor insert that contains the reaction mixture thereby elevating anypotential materially incompatibility issues. The present invention is anextension of the above-referenced integrated microfabricated reactor ofabove-referenced copending application Ser. No. 07/938,106 and theabove-referenced optically integrated micro-reaction chamber ofabove-referenced copending application Ser. No. 08/489,819. The sleevereaction chamber can be utilized in chemical reaction systems forsynthesis or processing of organic, inorganic, or biochemical reactions,such as the polymerase chain reaction (PCR) and/or other DNA reactions(such as the ligose chain reaction), or other synthetic,thermal-cycling-based reactions.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a partial cut-away perspective view of a microfabricatedchemical reaction instrument mounted in a power source/controlapparatus.

FIG. 2 is a schematic of the reaction instrument of FIG. 1.

FIG. 3 schematically illustrates a heating and detection arrangement fora microfabricated reaction chamber.

FIG. 4 illustrates an embodiment of a microfabricated silicon-basedsleeve reaction chamber made in accordance with the present invention.

FIG. 5 is an array of the sleeve reaction chambers of FIG. 4 operativelyconnected to a microelectrophoresis array.

FIG. 6 is an enlarged end view of another embodiment of a sleevemicroreaction chamber similar to FIG. 4.

FIG. 7 illustrates in cross-section embodiment of an enlarged section ofFIG. 6 using an isolated heater version, fixed window.

FIG. 8 illustrates in cross-section another embodiment of the sameenlarged section of FIG. 6 using a non-isolated heater version variablewindow.

FIG. 9 is a view of a hand-held instrument (PCR man) which utilizes thereaction chambers of FIG. 6 as inserts to change reactions.

FIGS. 10A and 10B illustrate a thermal cycling instrument utilizingseveral hundreds of individually-controlled silicon-based microreactionchambers.

FIG. 11 illustrates a schematic representation of high-throughput DNAamplification, sample-handling, and electrophoresis system.

FIG. 12 is an embodiment of an insert/lining for a reaction chamber withoptical window, with the top/cover open.

FIG. 13 illustrates external filling of a reaction chamber insert/liner.

FIG. 14 illustrates immobilized reagents/probes for detection ofspecific products directly on windows or within reaction fluid a s “teststrip” detected optically in the hand held instrument (PCR man) of FIG.9.

FIGS. 15 and 16 schematically illustrate optical detection systems foruse with the microreaction chambers of FIG. 6.

FIG. 17 schematically illustrates the use of integrated detection for anartificial intelligent feedback system.

FIG. 18 is a diagram showing the electrochemical oxidation and chemicalreduction reactions for tris (2,2′bipyridyl) ruthenium (II) (TBR) andtripropylamine (TPA).

FIG. 19 illustrates a method for tagging and separating DNA fordetection and quantification by electrochemiluminescence (ECL).

FIG. 20 illustrates cell voltage and ECL intensity versus time, with thevoltage being increased, then decreased.

FIG. 21 illustrates an embodiment of a micromachined ECL cell with athin film anode, and an associated photodiode detector.

FIG. 22 is an enlarged cross-sectional view of the ECL cell of FIG. 21with the electrical leads.

FIGS. 23-30 illustrate the fabrication process for producing an ECLcell, as illustrated in FIG. 21.

FIG. 31 illustrates an embodiment using Al on ITO on glass which reducesresistance of the ITO electrode.

FIGS. 32A and 32B show linear and parallel multiple series ofsleeve-type reaction chambers along a series of interconnecting tubeswith reaction control, detection, and logic feedback units.

FIGS. 33A and 33B show embodiments of a reaction control unit thatutilizes a micromachined (i.e., sputter-deposited) magnetic film tocontrol magnetic particles in the reaction chamber.

FIGS. 34 illustrates the integration of a reaction control element in amonolithic flow-through system.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is a micro-fabricated sleeve chemical reactionchamber. A silicon-based embodiment combines features such as dopedpolysilicon for heating and bulk silicon for conventive cooling, forexample. The microreaction chambers, made of silicon-based ornon-silicon-based materials can be used in an array for ahigh-throughput microreaction unit, or in a hand-held unit. The reactionchambers may combine critical ratios of non-silicon and silicon basedmaterials to provide the thermal properties desired. The hereinafterdescribed silicon-based embodiment combines, for example, a criticalratio of silicon and silicon nitride to the volume of material to beheated (e.g., liquid) in order to provide uniform heating, yet low powerrequirements. The invention also will allow the introduction of aninsert or secondary tube (e.g., plastic) into the reaction sleeve thatcontains the reaction mixture thereby alleviating any potentialmaterials incompatibility issues. The hereinafter described embodimentof the present invention utilizes a particular geometry of silicon-basedmicro-reactors that have been shown to be very efficient in terms ofpower and temperature uniformity. The particular embodiment of themicrofabricated reactor described has been experimentally used as athermal cycling instrument for use in the polymerase chain reaction(PCR) and other chemical reactions, and has shown to be superior topresent commercial instruments on thermally-driven chemical reactors.The silicon-based or non-silicon-based sleeve reaction chamber of thisinvention can be utilized in place of the reaction chamber of themicrofabricated system of above-referenced copending application Ser.No. 07/938,106; and can be utilized with the integrated heater anddetection arrangement of above-referenced copending application Ser. No.08/489,819, and thus constitutes an extension of the microfabricatedchemical reaction systems in these copending applications.

To provide an understanding of a microfabricated chemical reactioninstrument and the integrated heating/detection arrangement, prior tothe description of the embodiment of the silicon-based sleeve reactionchamber made in accordance with the present invention, a description isset forth of a microfabricated chemical reactor and an integratedheating/detection arrangement of the two-referenced copendingapplications. While the sleeve reaction chamber is described hereinafteras being constructed of silicon-based materials, non-silicon-basedmaterial may be utilized for certain applications wherein the materialsare compatible with the reaction fluids and/or are chemically inert.

FIG. 1 illustrates an embodiment of a microfabricated chemical reactioninstrument generally indicated at 10, shown above a recessed sectionthereof, indicated generally at 11, in a power source/control system ofthe microfabricated reaction instrument, generally indicated at 12. Ahypodermic needle 13 is shown inserting a sample through a siliconerubber window 14 into the reaction instrument 10. The reaction iscontrolled and powered by: induction coupling, such as that between coilL_(CL) in the instrument 10 and a magnetic coil 15; by capacitivecoupling, such as that between the plates of capacitor C₃ and plates 16and 17; and by electromagnetic coupling between a resonant circuit, seeFIG. 2, in instrument 10 and a radio frequency antenna 18.

A schematic of the instrument 10 of FIG. 1 is illustrated in FIG. 2, andcomprises three reagent chambers 19, 20 and 21, which, for example, maycontain the DNA primers, the polymerase, and the nucleotides and anydetection-tag molecules, such as magnetic beads. The target DNA moleculeis placed in reagent chamber 19 by insertion of a hypodermic needle 13(FIG. 1) or the like through a silicone rubber or other type materialwindow 14. The reactants chambers 19, 20 and 21 are respectivelyconnected by channels 22, 23, and 24, having narrow midsections, notshown, to a reaction chamber 25. Typically the chambers 19-21 and 25have a volume ranging from microliter to nanoliters. The channels 22-24are equipped with Lamb-wave pumps LW₁, LW₂ and LW₃, respectively, forpumping reactants in chambers 19-21 through channels 22-24 in thedirection of the arrows into reaction chamber 25. The Lamb-wave pumpsmay be located on any wall, or on multiple walls, of the channels 22-24.The Lamb-wave pumps LW₁, LW₂, and LW₃ are connected respectively tocapacitors C₁, C₂, and C₃. The surface tension across the narrowmidsections of the channels 22-24 prevents the reactants in chambers19-21 from flowing into reaction chamber 25 until pumping is initiated.The inner surfaces of the channels 22-24 may be treated to raise thesurface tension thereby further inhibiting flow of the reagents when theLamb-wave pumps are not activated.

The reaction chamber 25 may be equipped with a Lamb-wave transducerLW_(C) and a heater H_(C). The Lamb-wave transducer LW_(C) is connectedto inductor L_(CL) (also shown in FIG. 1). The heater H_(C) is connectedto a resonant circuit consisting of an inductor L_(CH) and a capacitorC_(CH). The Lamb-wave transducer LW_(C) acts as an agitator, mixer, orsonochemical inducer, as indicated by the connected arrows 26 in chamber25.

A channel 27 connects the reaction chamber 25 to a detection chamber 28.The channel 27 is equipped with a Lamb-wave pump LW_(DP), which isconnected to a resonant circuit consisting of an inductor L_(DP) and acapacitor C_(DP). The detection chamber 28 is equipped with a Lamb-wavesensor LW_(D), which is connected to a capacitor C_(D).

Lamb-wave transducers have high mechanical Q values and can therefore bepowered by only a narrow range of alternating voltage frequencies. TheLamb-wave pumps (LW₁, LW₂, LW₃) and Lamb-wave sensor (LW_(D)) arepowered capacitively by generating an electric field between the plates(such as plates 16 and 17 of FIG. 1 for example) at the resonantfrequencies of the Lamb-wave transducers (LW₁, LW₂, LW₃, and LW_(D)).But, because the transducers have high Q values, only when the frequencyof the imposed field is near the resonant frequency of a transducer dothe transducer vibrate with any substantial magnitude. Similarly, theLamb-wave mixing chamber transducer LW_(C) is provided by an alternatingfrequency magnetic field generated by the coil (15 in FIG. 1) at themechanical resonant frequency of the transducer LW_(C). The heater H_(C)and the Lamb-wave pump LW_(DP) are activated by directing anelectromagnetic wave from the antenna (18 in FIG. 1) to the resonantcircuit C_(CH) and L_(CH), and resonant circuit C_(DP) and L_(DP),respectively. The frequency of the incident electromagnetic radiationmust correspond to the mechanical resonant frequency of the transducerLW_(DP), to activate the pump LW_(DP). The frequency of the incidentelectromagnetic radiation must correspond to the resonant frequency ofthe electrical elements CH, L_(CH) and H_(C) to activate the heaterH_(C).

A PCR reaction, for example, is initiated by pumping the reagents in thechamber 19, 20 and 21 along the directions of the arrows throughrespective channels 22, 23 and 24 to the reaction chamber 25 byactivating pump LW₁, LW₂, and LW₃. A series of about twenty to fortythermal cycles, for example, are then initiated, and during each cyclethe temperature of the reactants in the reaction chamber 25 goes from55° C. to 96° C., and back to 55° C., for example. The temperature ofthe reaction chamber 25 is determined by the power of the incidentelectromagnetic signal at the frequency corresponding to the resonantfrequency of the circuit composed of the capacitor C_(CH), and theinductor L_(CH), together with the heater H_(C). The Lamb-wave deviceLW_(C) of the reaction chamber 25 acts as an agitator or mixer, asindicated by arrows 26, to mix the reagents and promote the reaction.

When the thermal cycling is complete, the contents of the reactionchamber 25 are pumped by the Lamb-wave perm LW_(DP) through channel 27in the direction of the arrow to the detection chamber 38, whichutilizes a Lamb-wave sensor LW_(D). Alternatively, the detection chamber28 may be provided with an optical window and testing may be performedby fluorescence-based or absorption-based optical spectroscopy.

FIG. 3 illustrates a heating/detection arrangement that can beincorporated into the microfabricated reactor of FIGS. 1 and 2. As shownin FIG. 3, a chemical reaction chamber, such as a PCR chamber, of aminiaturized, microfabricated instrument, generally indicated 30, isillustrated in cross-section, with chamber 31 being formed in a housing32, constructed of Pyrex for example, and having silicon inserts 33 and34 therein, with an inlet 35 and an outlet 36. Energy from two differentenergy (light) sources is directed onto the housing 32, one source 37being infrared (IR) source, and the second source 38 being anultra-violet (UV) source. The IR source 17 applies heat more uniformlythrough the bulk of the solution in chamber 31. The UV source 18 inducesfluorescence of the reaction products in the visible (Vis) spectrum,which can be detected by a visible (Vis) detector 39 located external ofthe housing 32 defining reaction chamber 31. Housing 32 must beconstructed of a material transparent to UV and/or the visible spectrum.By incorporating an integrated excitation (heating) and detection systemin the reaction chamber itself, confirmation of the presence of a samplein the reaction chamber can be confirmed, and the dual reaction anddetection chambers 25 and 28 of the microfabricated reactor of FIG. 2can be consolidated, thus reducing fabrication costs by reducingcomponents.

The present invention, an embodiment of which is illustrated generallyin FIGS. 4 and 6 involves a microfabricated reactor generally indicatedat 40 which includes a silicon-based sleeve as a chemical reactionchamber, generally indicated at 41, constructed of two bonded siliconparts, and which utilizes doped polysilicon for heating and bulk siliconfor convective cooling, as described in greater detail hereinafter. Thesleeve 41 includes a slot or opening 42 into which reaction fluid,indicated at 43, from a hypodermic needle 44 is inserted into thereaction chamber, or into which a secondary tube 45 containing areaction mixture 46 may be inserted. The tube 45 is constructed ofplastic, for example, or other material which is inert with respect tothe reaction mixture, thereby alleviating any potential materialincompatibility issues. The sleeve is also provided with an opening 47in which is located an optical window 48, made, for example, of siliconnitride, silicon dioxide, or polymers. The silicon sleeve reactionchamber 41 includes doped polysilicon for heating and bulk silicon forconvective cooling, and combines a critical ratio of silicon and siliconnitride to the volume of material to be heated (e.g., liquid) in orderto provide uniform heating, yet low power requirements.

FIG. 6 is an enlarged view of microreaction chamber, similar to the FIG.4 embodiment, but utilizing two windows. The reaction chamber of FIG. 6,generally indicated at 50, is composed of two silicon wafers orsubstrates 51 and 52 bonded together as indicated at 53, and configuredto define a slot or opening 54 therein. Each of wafers 51 and 52 includea layer of silicon nitride 51′ and 52′ which define a window, indicatedgenerally at 55 and 56, respectively. Window 55 in wafer 51, constructedof silicon nitride, is provided with a heater 57 having electrical leads58 and contacts 59 which extend along the edges of heater 57 to provideuniform heating. Window 56 in wafer 52 has a heater not shown in FIG. 6but which is secured by metal contacts 60 and 61 as illustrated ineither of FIGS. 7 and 8. The silicon nitride layers 51′ and 52′ are verythin (about 1 μm) and vapor-deposited onto the bulk silicon wafers 51and 52. The silicon nitride only becomes a window, as indicated at 55and 56, when the bulk silicon wafers 51 and 52 are etched away to formthe opening or slot 54. Heater 57 is transparent to energy passingthrough window 55, for example.

FIG. 7 is a greatly enlarged view of an embodiment of a section ofsilicon wafer 52 and window 56, as indicated by the circle 62 in FIG. 6.As seen in FIG. 7, the section of the silicon wafer 52, indicated at 63,is composed of bulk or single crystal silicon and is in contact with alow (100 to 500 MPa) stress silicon nitride membrane or window 64 (52′in FIG. 6) which in turn is in contact with a doped polysilicon heater65 and metal contact 60 and 61. The FIG. 7 embodiment comprises anisolated heater version fixed window.

FIG. 8 is a greatly enlarged view of another embodiment of a section ofsilicon wafer 52 and window 56, as indicated by the circle 62. As seenin FIG. 8, the sections of the silicon substrate 52, indicated at 66 arecomposed of bulk or single crystal silicon. As in the FIG. 7 embodiment,a low (100 to 500 MPa) stress silicon nitride member or window 69 (52′in FIG. 6) is in contact with silicon section 66, a doped polysiliconheater 70 is in contact with window membrane 69 and metal contacts 71are mounted to heater 70. The FIG. 8 embodiment comprises a non-isolatedheater version. The window size relative to the chamber can be varied toensure thermal uniformity and optical access to the reaction chamber.

By way of example, the silicon wafers or substrates 51 and 52 may have alength of 5 to 50 mm, width of 2 to 10 mm, thickness of 0.1 to 1.0 mm,with the slot 54 having a cross-sectional area of 5 to 500 mm². Slot 54,which shown to be of a six-sided configuration, may be a round, oblong,square, rectangular, or other configuration. Windows 55 and 56 may havea length of 0.1 to 1 mm, width of 0.1 to 50 mm, thickness of 0.1 to 10μm, and in addition to silicon nitride, may be composed of silicondioxide, silicon, or polymers. The doped polysilicon heater 65 of FIG. 7may have a thickness of 0.05 to 5 μm, with the heater 70 of FIG. 8having a thickness of 0.05 to 5 μm. The metal contacts 60-61 and 61′ ofFIGS. 6 and 7 may be composed of gold or aluminum, with a thickness of0.01 to 5 μm, with the metal contact 71 of Figure having a thickness of0.01 to 5 μm and composed of gold or aluminum. The heater 57 in siliconwafer or substrate 51 is composed of doped polysilicon having athickness of 0.05 to 5 μm, with the electrical leads and contacts 58 and59 being composed of gold or aluminum.

The use of bulk silicon, polysilicon, silicon nitride enablesflexibility in design for thermal and optical properties of eachchamber. This enables individually controlled, thermally isolatedreaction chambers in a small instrument (FIG. 9) or in large instrument(FIG. 10).

FIG. 9 is an embodiment of a miniature thermal cycling, batteryoperated, hand-held low-power, feedback-controlled instrument for PCRthat uses microfabricated, silicon-based reaction chambers, such asthose of FIGS. 4 and 6, the development of which addressed thermaluniformity and temperature precision of the reaction chambers,temperature ramp rates of the chambers, and biocompatibility of thematerials in contact with the reagents.

As shown in FIG. 9, the hand-held, battery-operated instrument, coined“PCR man”, generally indicated at 75, comprises a pressure-fitelectrical contact controller holder, or housing 76, which for examplemay be 3×5 inches having a control-face-plate 77 with various indicatorsthereon, including a “status” window 78. The holder 76 is provided witha thermocouple-based temperature feedback control circuitry, heaterelectronics, computer interface, and power source connector, asdescribed in greater detail hereinafter. The holder 76 is provided withbatteries, indicated at 79, such as four nine-volt batteries, and at theupper end is provided with slots 80 for insertion of reaction chambersinside the holder (three slots shown), and into which silicon-basedreaction chambers 81, 82, 83 and 84, with integrated heaters (as shownin FIG. 6) are inserted as indicated by the arrow 85. The reactionchambers 81-84 may when constructed contain different reagents orchemicals, and can be selectively inserted into the hand-held instrument75 via slots 80 in holder or controller 76.

This instrument can be used to rapidly and repetitively providecontrolled thermal cycles to the reaction mixture. The thermalconductivity properties of the silicon or similar semiconductingsubstrate, for example, help speed up the thermal rise and fall times,and allow low power operation. While silicon is unique in its thermalproperties, i.e., high thermal conductivity, a combination of silicon,silicon nitride, silicon dioxide, polymers and other materials wouldprovide a combination of thermal conductivity and insulation that wouldallow thermal uniformity and low power operation.

While silicon or silicon-based materials is preferable, other materialscan be used, including: polymers, ceramics (crystalline andnon-crystalline, silicate and non-silicate-based), metals or combinationof metals (alloys), composites, and a combination of materials (such ascomposite polymers that contain dopants (for example, aluminum oxide)that increase thermal conductivity, for example) in order to achieve thedesired thermal properties (conductivity, resistance, specific heat,expansion, etc.), thermal mass, or other sensing and controlcapabilities. The compatibility of such materials need to be consideredas well, especially in regards to its surface reactivity or inertness.The materials should also be selected based upon the ability orcapability to integrate control elements onto or adjacent to them. Inthe case where liners are used, the chemical compatibility of lessimportance, but other features such as conductivity, etc., may still beof critical importance. For example, it is possible to make a reactionchamber out of the proper thermal material (highly conductive) such assilicon or metal (i.e., copper) which may be noncompatible with thereaction, and use a chemical vapor deposition or evaporation process todeposit an ultra-thin polymer passivation layer (such as teflon orpolypropylene) on the walls to achieve a compatible surface that isminimally compromised in terms of thermal conductivity.

It is the use of such microfabrication technologies that allows for suchunique capabilities. Without them it would be difficult to fabricatereaction chambers with optimal reaction-control capabilities. Thin filmprocesses and vapor deposition, for example, allows for extremely thinand uniform coatings of materials onto other materials, while etchingand batch-fabrication allow for mass production.

Integration of control elements onto this variety of materials, whichmay include deposition (or other microfabrication method), is nowpossible in one form or another. For example, thin film metal heaterscan be deposited onto polymer or ceramic devices, as can electrodes,sensing elements, circuits, etc. The formation of IC-type electronicsand control elements can be deposited onto many of the materialsavailable today. The selection of the materials and microfabricationmethod only needs to be based on the needs of the application, since awide variety of options exist. Reaction chambers are a prime example ofan application that benefits from this breadth of availability. Theheating and/or cooling means for the reaction chamber may be composed ofthermo electric film, for example.

Integration of microfabricated magnetically-active films, for example,with the reaction chambers or series of reaction chambers can be used tocontrol reaction reagents and products. This can be accomplished by theuse of magnetic or paramagnetic particles in the reaction fluid that arecapable of selectively binding to specified reagents. Through the use ofthe magnetic attraction and repulsion forces that can be created betweenthe film and the particles, the desired reagents could be selectivelyattracted or repulsed, while the retaining reagents could be carriedaway via the flow-through system.

The incorporation of microfabricated thermoelectric films or heaters, asanother example, could be used to control the temperature of thereactions in the sleeve-type reaction chambers. Both heating and coolingcan be accomplished with such films allowing the reagents to experienceelevated and below ambient thermal regimes. Series of temperature ormechanically actuated pressure zones will generate fluid flow conditionsfor directed throughput.

Other active micromachined films or devices can be incorporated toaffect the reaction rate, mixing, flow or transport within the system.Examples include microfabricated actuators such as shape memory films(i.e., NiTiCu), electrostatic actuators such as polyimide, thermalbimorph actuators such as polyimides or other materials with differentthermal expansion coefficients, and other micromachined structures.

The particular embodiment, such as FIG. 6, of a microfabricated reactordescribed can be used as a thermal cycling instrumentation for use inthe PCR and other chemical reactions, biochemical processes,microbiological processes, and incubators. As shown hereinafter thereaction chamber of this invention is superior to present commercialinstruments used in thermally-driven chemical reactions.

During the experimental verification of the instrument of FIG. 9 and themicroreaction chambers for use therein, such as illustrated in FIGS. 4and 6, several different sizes of PCR reaction chamber designs werefabricated using integrated circuit (IC)-type silicon processing steps.The generalized fabrication process was as follows: Three-inch round,0.5 mm thick single crystal silicon (SCS) wafers were processed in thefollowing way: low stress (200-300 MPa) silicon nitride (Si_(x)N_(y))was low-pressure chemical vapor (LPCVD) deposited onto entire wafer(1.0-2.0 μm thick). Photolithographic patterns for reaction chamber andsubsequent processing steps were taken in the following order: 1) thesilicon nitride was reactive ion etched (RIE) over the reaction chamberarea, 2) the SCS was etched to the silicon nitride backside defining thechamber volume, 3) the wafer was patterned and the silicon nitride ischemically etched away everywhere except over the nitride membrane orleft over the entire surface, depending upon the reaction chamberdesign, 4) the remaining silicon nitride membrane (side opposite thechamber) was LPCVD deposited with polycrystalline silicon (polysilicon)to a thickness of 3000Å, 5) the polysilicon was then high temperaturedoped with boron to a resistivity of 50-200 ohms per square, and 6)either aluminum or gold thin-film metal contacts were deposited definingthe heater geometry.

Each wafer potentially contains many reaction chambers, depending upongeometry and volume desired. The etched depression in each waferconstitutes one-half of a dual-heater reaction chamber. Processed wafersare subsequently bound together forming an enclosed chamber with heaterson both sides.

The reaction chambers can be bonded together by depositing a thin filmof low-temperature-curing polyimide between the two wafers directly orother bonding techniques such as eutectic metal bonding. A highprecision computer-controlled silicon saw was used in each design to cutout each dual-heater chamber. The chambers were then rinsed repeatedlywith de-ionized water and dried prior to treatment with silane.

The reaction chambers were inserted into a pressure-fit electricalcontact holder that was part of the plexiglass backboard of theelectronics components making up the controller. The controllerelectronics could be either/or analog or digital and could use processessuch as pulse-width modulation as a feedback control mechanism. Thebackboard was 3 inches by 5 inches and consisted of thethermocouple-based temperature feedback control circuitry, heaterelectronics, computer interface, and power source connector. Thecircuitry was designed to work from 8 to 32 volts. Thermal calibrationwas accomplished by correlating the temperature of the fluid with thatof the silicon-measuring Type K thermocouple. Once calibrated, theinstrument was capable of automated, feedback-controlled, thermalcycling operation without direct measurement of the reaction fluid. Thethermal cycler output is to an Apple Centris 650 computer which displaysthe thermal cycle real-time along with storing the accumulated profiles.Four nine-volt batteries were able to run the entire instrumentcontinuously for over 2.5 hours.

Typical PCRs were set up as scaled-up master mixes, to assure uniformitybetween aliquots thermocycled under different conditions. Reagentamounts were based on those ideal for 50 ul reactions. In general,master mixes contained: 50 mM KCl, 10 mM Tris-HCl pH 8.3, 1.5-3.0 mMMgCl₂, 200 uM each deoxynucleotide, or 800 uM dNTP total, 0.5 uM each oftwo oligonucleotide primers, 25 units/ml AmpliTaq® DNA polymerase, andtarget template at a specified copy number per 50 ul reaction. Templatefor some of the β-globin PCRs was added as single strand DNA from a M13bacteriophage clone of a portion of the human β-globin gene. CF templatewas human genomic, double stranded, DNA derived from a cultured celllines, HL60, GM07460, or GM08345. Each reaction mixture was aliquotedfrom the same master mix and thermocycled in the instrument of thepresent invention and a Perkin-Elmer GeneAmp® 9600 Thermal Cycler.Thermocycled reactions from both thermal cyclers were fractionated on 3%NuSeive, 1% Seakem agarose (FMC Corp.) using tris-borate buffer. Thegels were stained with ethidium bromide and photographed underillumination with 302 nm UV light.

Although initially conceived as a single use, disposable reactionchamber, the robust nature and stable properties allowed for repeateduse of the reaction chambers.

The (MEMS) based thermal cycling instrument of this invention has beentested with a variety of PCR systems, including viral, bacterial, andhuman genomic templates. As well, various changes in both the reactionchamber design and controller instrumentation have been implemented andevaluated. A controller output real-time display of a thermal cycle frommicrofabricated thermal cycler has been prepared and it has been shownthat with 15 volts input (average 1.2 Watts) that heating rates of over5° C./sec are attained. Cooling is slightly slower (2.5° C./sec.) mostlydue to the fact that the reaction chamber is held inside a Plexiglasinstrument board. Precision of +/−0.5° C. is maintained at the targettemperatures. Higher heating and cooling rates have been achieved.

We have performed experiments that show the quantitative nature of thePCR process in both FIG. 9 and commercial instruments. These experimentsconsisted of removing 5 μL aliquots out of a 105 starting copies,β-globin PCR from both the instruments at 23, 25, 27, 29, and 31 cycles.These aliquots were subsequently run on an agarose electrophoresis gel.The results from both instruments are virtually identical. The samequantitative gel electrophoresis series results from the amplificationof the 268-bp target of β-globin directly from human genomic (HL60) DNAwere performed.

Multiplex PCR is considered to one of the most recent andanalytically-powerful DNA amplification techniques. It requires preciseand uniform temperature control within the reaction chamber. We haveachieved this with the instrument of this invention.

Post-PCR-detection of the specific mutations associated with the cysticfibrosis (CF) disease, for example, can be identified with simplenylon-based test strips, using reverse-dot-blot technology. The teststrip has specific, immobilized DNA probes containing the mutationsequence of interest. The multiplex PCR amplification products are putinto a simple reagent trough along with the assay. If binding occurs andthe DNA is retained after a wash step, theDNA-biotin-streptavidin-enzyme complex will turn color upon treatmentwith the substrate. The commercial and the FIG. 9 instrument-amplifiedresults of PCR followed by reverse-dot-plot assay for CF prepared.

From the results of the above-referenced experiments and previousresults, relative to the above-identified copending applications, withsingle-sided heaters, silicon-based reaction chambers of various sizesand configurations are capable of carrying out chemical reactions, suchas PCR, with low power requirements.

The significance of the above-reference experimental results is that forthe first time, battery-operated, hand-held, PCR amplification; andsimple reagent-based, targeted detection of complex biologicals anddiseases can be carried out in an instrument such as illustrated in FIG.9.

The rapid temperature cycling and thermal uniformity now possible in aPCR-type compatible silicon-based microreaction chamber may provideinsight into hybridization and enzyme kinetics. For example, theimportance of temperature control is paramount in the PCP process,especially when complex systems are to be amplified (e.g., human genomicDNA, multiplex amplifications). Precise temperature control as well asthermal uniformity must be balanced. To truly miniaturize the instrumentor take advantage of microfabricated reaction chambers in order to builda high-throughput instrumentation, such as illustrated in FIGS. 10A, 10Band 11, one must integrate the control elements on a unit-by-unit scale.Thermal properties of the various materials used must also be balancedto combine efficient control with thermal liability. Silicon-basedmaterials afford the requisite thermal properties, the ability tointegrate heaters and feedback control, and their manufacture takesadvantage of highly parallel, automated, and batched processing.

FIGS. 10A-10B and 11 illustrate a system approach, combining thehigh-throughput, high efficiency thermal cycler instrument, samplehandling, and electrophoresis modul. The electrophoresis module couldalso be micromachined in glass or silicon. The instrument could behybrid in nature; i.e., a silicon based reaction chamber and a miniglass electrophoresis module taking advantage of both substrates ormembers, as in the FIG. 5 embodiment. The advantage to having real-timedetection of DNA production is that it allows the operator to know aboutthe PCR efficiency during the reaction, rather than waiting to see theresults on a gel. This will significantly help DNA sequencingproductivity by eliminating time wasted running electrophoresis gels onsamples that haven't amplified.

FIGS. 10A and 10B illustrate a thermal cycling instrument, generallyindicated at 90, having a housing 91 with a face plate 92 with variousindicators thereon, including a “status” window 93, similar to thefaceplate of the FIG. 9 hand-held instrument. The housing includes ahinged top 94, under which is located an array 95 (see FIG. 10B) ofindividually controlled silicon-based microreaction chambers 96, whichmay, for example, be of the type illustrated in FIGS. 4 and 6. Theinstrument 90 is designed for 384 microreaction chambers 95, althoughthe array 95 as shown in FIG. 10B only includes 100 chambers forsimplicity of illustration.

FIG. 11 is a schematic representation of high-throughput DNAapplication, sample-handling, and electrosystem utilizing the instrumentof FIGS. 10A-10B, and corresponding reference numeral indicatecorresponding components. An array 95′ of 384 individual-controlled PCRreaction chambers 96′ (only five shown, is operatively connected to anautomated sample input/output assembly, generally indicated at 97 usingtwo sets of microinjectors, generally indicated at 98 and 99. The sampleinput/output function between microinjector set 98 of assembly 97 andarray 95 is indicated by double arrow 100, while the function betweenthe sets 98 and 99 of microinjectors is indicated by double arrow 101.The microinjector set 99 is operatively to an array 102 of individualmicroelectrophoresis channels 103. This injector input/output systemwill load reagent samples from the reaction chambers 96 with vacuum orelectrokinetic power; automatically or robotically move toelectrophoresis channels 103; and unload reagents via pressure orreversed field electrokinetic injection into those channels forelectrophoretic separation. The electrophoresis module could bemicromachined as well. Silicon is good for reaction chambers, glass forelectrophoresis.

The electrophoresis channels 103, formed in a glass substrate are eachdirectly connected to a silicon reaction chamber of the type shown inFIG. 4, so as to produce an array 95 of reaction chambers 96′ connecteddirectly to the array 102 of electrophoresis channels 103, as shown inFIG. 5.

By use of material in addition to silicon and silicon-based materialdescribed above with respect to FIGS. 5 and 11, the PCR/electrophoresiscapability can be expanded. For example, the reaction chamber can besilicon, metal, ceramic as above for thermal considerations, the linercan be a polymer, glass, or other appropriate for compatibility andcould have micromachined (CVD or evaporated) layers, and theelectrophoresis channels could be glass, polymer, ceramic for electronicinsulation or electronic in general considerations (this also could haveCVD etc. deposited layers for compatibility and for control ofelectroosmotic flow or zetra potential). All the same concepts hold fromabove (i.e., IC-type integration of control elements. For example, aseries of electrodes and optical detectors could be fabricated directlyonto the substrate. The liner in that case actually directly interfacesto the electrophoresis microchannels.

Removable/permanent liners/inserts for the reaction chambers of amaterial known to be compatible with the appropriate reactions, such asshown in FIG. 12 will in some applications reduce the overall cost, asthese liners/inserts may be disposable. Also, considered arederivatizing agents for the surfaces of the silicon-based reactionchamber to enhance covalent and/or other bonding to the liners. Examplesbeing the organic/reactive silanes, polyimides, teflons, polytheylene,other polymers.

FIG. 12 illustrates an embodiment of an insert/liner, generallyindicated at 105, for a reaction chamber with an optical window 106therein. The insert/liner 105 includes a six-sided housing 107 and atop/cover 108. The six-sided housing 107 is configured, for example, tobe inserted into opening 54 of the reaction chamber 50 of the FIG. 6embodiment, such that window 106 aligns with one of windows 55 or 56 ofFIG. 6. The housing 107 may be constructed of plastic or othercompatible material set forth above. Window 106 of insert/liner 105includes a test strip 109, described hereinafter with respect to FIG.14.

FIG. 13 illustrates external filling of the reaction chamberinsert/liner 105 of FIG. 12 via an external interfludic connection,generally indicated at 110. Examples of fluidic connections includes:syringe needles, pipette tips, and fused silica capillaries or glass orpolymer tubing.

Surface immobilization of the windows (or test strip) with probes foroptical or other detection (other microbased detections) of productproduction and specificity, can be provided as shown in FIG. 14 which isan enlarged view of the test strip 109 of FIG. 12. Such a test strip canbe included in the windows of the FIGS. 4 or 6 reaction chambers.Immobilized reagents/probes for detection of specific products directlyon the window, such as 106 of FIG. 12, or within the reaction fluid inreaction chamber insert/liner 105 of FIG. 12, can be detected opticallyin the PCR man hand-held instrument of FIG. 9, by the use of the teststrip 109. The actual inner surface of the window could be used as animmobilization surface for specific-target or product detecting probes,or the window could be used to view an immobilization/detection surfacewithin the chamber.

FIGS. 15 and 16 schematically illustrate two setups for opticaldetection. The FIG. 15 setup is a laser/ccd version, while the FIG. 16setup will allow low-power operation for implementation into the PCR man(hand-held instrument) of FIG. 9.

As shown in FIG. 15, this optical detection arrangement for a reactionchamber 120 with a window 121 and control electronics 122, includes anoptical filter 123, such as an interference filter or band pass filterfor passing the detection wavelength of interest, CCD 124, digitizedimage generally indicated at 125, focusing optics 126,reflector/splitter 127 and an Argon ion laser 128. The operation is asfollows: The laser excites the fluorescent indicator dye associated withproduct detection. The fluorescent signal is monitored by the CCD 124.Absorption spectroscopy could similarly be used.

FIG. 16 is a miniaturized optical detector system for reaction chamber120′ having a window 121′ and control electronics 122′ is composed oftwo filters 130 and 131, a solid state detector 132 and a Blue LED 133.The filters 130 and 131 are either band pass or long pass for selectingemission (i.e., 600 nm long pass) and band pass for selecting theexcitation wavelength of interest, such as 488 nm±10 nm. The excitationband pass can be used to select from the typically broad emission of anLED, for example. The operation of the FIG. 16 detection system is asfollows: The LED is filtered to 488±10 nm as an excitation source (orabsorption) for the fluorescent indicating dye. The solid state detectoris also filtered to receive only the wavelengths of detection (>600 nm)or as an absorption detector.

Artificial intelligence is one way to produce DNA and determine how manycycles to go, when it is complete, if it worked, adjustment ofparameters to improve production, etc. Using a real-time detectionsystems such as illustrated schematically in FIG. 17, an artificialintelligent feedback system using integrated detection can be provided.The system of FIG. 17 comprises a reaction chamber 135 having a window136, a detector 137 for in situ detection of DNA production, aninstrument control 138 for reaction chamber 135, and a data readoutsystem 139, which receives data from detector 137, as indicated by arrow140, and supplies control data to controller 138, as indicated by arrow141. The data readout system 139 provides information such as how muchDNA is being made, starting copy number, reaction complete, etc. Byquantifying the DNA production via the optical monitoring system, whichis well known, the system could adjust its cycling time and cycle numberto produce the minimal number of cycles required for detection, thusspeeding up the process. Also by determining the cycle number requiredto detect a given fluorescent signal, or product concentration, thesystem would be able to calculate all starting copy number orconcentration of the unknown starting sample. This would allow automatedconcentration calculations. Real-time quantitative information can allowthe system to adjust the reaction parameters such as targettemperatures, hold times, and ramp rates.

A microfabricated, electrochemiluminesence cell for the detection ofamplified DNA is described hereinafter with respect to FIGS. 18-31, andwhich sets forth the design, fabrication, and testing thereof. Themicrocell is designed to be the detection unit in a PCRmicro-instrument, such as described above and illustrated in FIG. 9. Thecell is a vertical assembly of micromachined silicon and glass andcontains thin film electrodes, as shown in the Figures.

The detection of DNA by means of electrochemiluminescence starts withDNA amplification by PCR, to increase the concentration to detectablelevels. Then it is labeled with tris (2,2′ bipyridyl) ruthenium (II)(TBR). Oxidized TBR luminesces (orange) upon reduction. Oxidation occurselectrochemically at an electrode surface, hence the light emission isreferred to as electrochemiluminescence (ECL). TBR requires a relativelylow oxidation potential (a few volts) and has a high ECL efficiency inthe visible (620 nm). This makes it attractive for microsensorapplications, since visible emission is readily detected with siliconphotodiodes, which could be integrated into a silicon micromachinedcell. The reduction can occur electrochemically or chemically; in eithercase, light is emitted. For example, oxidized tripropylamine (TPA)readily transfers an electron to oxidized TBR, whereupon the TBRchemiluminesces. Since both oxidations can occur at the same electrode,relatively large concentrations of both species can be produced in closeproximity, which results in higher light intensity for a given TBRconcentration, than if TBR alone is present in solution. Theelectrochemical oxidation and chemical reduction reactions for TBR whichoccurs at the anode are schematically diagrammed in FIG. 18.Electrochemical reduction of TBR also occurs at the cathode. In order tooxidize only the TBR labeled DNA and not the free TBR, a separation ofthe two is required. One way to achieve this is by using the highlyspecific binding of immunoproteins (antibody-antigen).

An example is shown in FIG. 19, where a biotin primer is made on a 5′end of one strand of target DNA and the TBR is tagged to the 5′ end ofthe complementary strand. During the PCR process DNA double strands areproduced with biotin and TBR labeled on either end. The biotin labeledDNA can then be introduced into an electrochemical cell with an anodewhose surface is coated with avidin, the antibody for bitoin. Selectivebinding will occur, after which the solution in the cell is flushed toremove any “free” TBR. Now the TBR, bound to the DNA, which in turn isattached to the anode via the antibody-antigen bond, can be oxidizedalong with added TPA, and the subsequent luminescence intensity willdepend on the amount of DNA that is present.

The ECL microcell, as described in greater detail hereinafter withrespect to FIGS. 21-31, is a multilayer assembly of micromachinedsilicon and glass. Cells with solution capacity ranging from 35 μL to 85μL have been designed and fabricated in silicon. An e-beam deposited,gold, thin film forms the cell cathode. The anode is also a thin film.Experiments with both indium tin oxide (ITO) and platinum have beencarried out. ITO is transparent to visible light, so that when depositedonto glass, it can form the top layer of the assembly, through which theemitted light can be picked up by a photodetector (see FIG. 21). Theassembly also contains micromachined fluid fill ports (see FIG. 22). Thelayers were assembled and bonded together (see FIGS. 29-30) using a lowtemperature curing polyimide, such as Epotek 400.

ECL experiments have been performed in the microcell with free TBR,i.e., no DNA. The cells were filled with TPA +TBR solution and aphotomultiplier tube (PMT) was placed in close proximity to the topglass layer of the cell to detect emission. The chemiluminescenceproduced by the reaction of oxidized TPA and TBR depends on theconcentration of both chemicals. In these experiments, the concentrationof TPA was kept constant (50 mM) and TBR was varied. The solutions wereprepared as follows: 1 g of TBR hexahydrate chloride was dissolved in 50mM TPA to make 5 mM of TBR, which was then diluted with additional 50 mMTPA to produce a set of test solutions, whose TBR concentrations rangefrom 0.1 nM to 5 mM. An EG&G potentiostat, model PARC 273, was used toproduce voltammograms of the TBR+TPA solution, both in the microcellwith ITO and gold thin film electrodes, and in a more conventional,electrochemical cell with platinum wire electrodes. From thevoltammogram, the oxidation potential, which is where ECL occurs, wasdetermined and then applied as a dc bias between the thin film cathodeand anode. The emitted light was measured with a Hamamatsu MT, modelR928, biased at 600 volt. FIG. 20 shows the relationship betweenmeasured light intensity and electrode voltage for a TBR concentrationof /mM, where cell voltage and ECL intensity versus time. The voltage,as indicated by the dot-dash-dot line, is increased, then decreased. Inboth directions, the voltage passes through the oxidation potential ofTBR, where intensity of ECL is a maximum. In tests conducted thus far,the lowest concentration of TBR that has been measured using themicrocell with an ITO film as the anode material was 1 μM. With aplatinum anode, the measured TBP concentrations were as low as 1 nM. Therelatively high resistance of the ITO film is believed to be limitingthe oxidation current for TPA, and therefore reducing the sensitivity.It has been determined that sensitivity can be improved by depositing athin film of material, such as aluminum on the ITO film, as describedhereinafter with respect to FIG. 31. Also, efforts are being carried outto integrate the silicon photodiode into the microcell, rather thanbeing separated therefrom as in the FIG. 21 embodiment.

FIG. 21 illustrates an embodiment of a micromachined ECL cell with thinfilm anode, generally indicated at 140, and a silicon (Si) photodiode141 positioned adjacent the ECL cell 140. The ECL cell 140 is shown inenlarged cross-section in FIG. 22. The cell 140 comprises a pair ofsilicon members 142 and 143, between which is positioned an electrode144, which may be constructed of gold (Au), platinum (Pt) or silver(Ag), an ITO layer 145, and a glass layer or slide 146. Silicon member142 includes a reaction chamber 147, and member 143 includes a pair offilling ports 148 (see FIG. 22) via which an analyte, as indicated bylegend is directed into chamber 147 and withdrawn therefrom via tubes orlines 149 and 150, as indicated by arrows 151 and 152. As seen in FIG.22, a center section 153 of silicon member 143 located between fillports 148, along with ITO layer 145 and glass slide 146 define a windowby which reactions within chamber 147 can be detected, as indicated byphotons 154 passing therethrough onto photodiode 141. Electrical leads155 and 156 are connected from a power source to electrode 144 and ITOlayer 145, respectively, while photodiode 141 is electrically connectedto a power source via leads 157 and 158.

FIGS. 23-30 illustrate the fabrication of an embodiment of an ECL cellsimilar to that of FIGS. 21 and 22. The fabrication process is carriedout as follows:

1. A block 160 of silicon is coated to form a layer 161 of siliconnitride (see FIG. 23).

2. A layer 162 of photoresist is deposited on the layer 161 (see FIG.24).

3. The layer 162 is patterned and photolithographic process to form anopening 163 therein (see FIG. 25).

4. The section 161′ of silicon nitride layer 161 beneath the opening 163is removed by RIE etching (see FIG. 26).

5. A section of silicon block 160 is removed by KOH etching to form areaction chamber 164, and the remaining photoresist 162 is removed (seeFIG. 27).

6. A layer of gold, for example, is deposited by thin film evaporationover the upper surface of block 160 and chamber 164 to form an electrode165 (see FIG. 28).

7. A second block of silicon 166 is coated with a layer 167 of siliconnitride and openings 168 and 169 are formed therein by RIE etching, anda pair of filling ports 170 and 171 are formed, as by micromachining, inblock, 166, and silicon nitride coated block 166 is bonded to electrode165 (see FIG. 29).

8. A layer of ITO forming an electrode 172 is deposited on a layer orslide 173 of glass, and then bonded to the silicon nitride layer 167(see FIG. 29).

9. Electrical leads 174 and 175 are secured to gold electrode 165 andITO electrode 172, a detector 176, such as the photodiode of FIG. 21,having electrical leads 177 and 178 is bonded to glass layer 173, andthe silicon nitride coated silicon block 160 is positioned on a magnet179 having electrical leads 180 and 181 (see FIG. 30).

To reduce resistance of the ITO electrode 172 a thin film of aluminum182 (see FIG. 31) can be deposited on the ITO layer or electrode 172prior to same being bonded to the silicon nitride coated silicon block166.

FIG. 32A illustrates a linear multiple series of sleeve-type reactionchambers 40′ interconnected by tubes 190 extending from an end of onechamber to an end of a next chamber, and having an energy coupler 191illustrated over only one of the reaction chambers 40′ to form, a“string of beads” flow through reaction system. In operation the energycoupler 191 would be positioned on reaction chamber 40′. FIG. 32Billustrates a linear and parallel multiple series of sleeve-typereaction chambers 40′ with interconnecting end to end tubes 190′ as inFIG. 32A and interconnecting tubes 192 extending between openings 193 inthe sides of the reaction chambers 40′. In FIG. 32B the reactionchambers are connected to a control, such as computer 194 and to adetector 195 to provide logic feedback as indicated by arrow 196, so asto produce a reaction element electronic control network indicated at197, with net flow being indicated by arrows 198, 199, 200 and 201. This“string-of-beads? flow through reaction system is made up ofinterchangeable reaction chamber elements on a flow-through tubingsystem.

FIGS. 33A and 33B illustrate an embodiment of a reaction control unitsthat utilize a micromachined (i.e., sputter-deposited) magnetic film tocontrol magnetic partides in the sleeve reaction chamber. Other examplesof micromachined elements are: electroactive films such as heaters,electrodes, thermoelectric and mechanical (i.e., shape memory) thinfilms, photodiodes, etc. The reaction chambers with integrated reactioncontrol or detection element are coupled to an external energy source.FIG. 33A illustrates an embodiment of a sleeve-type reaction chamber 240formed by two members 241 and 242 having an opening or slot 243 ofsix-sided configuration, with microfabricated control film 244 depositedthereon. A reaction chamber liner to tube connector 245 is connected inslot 243, and an energy coupler 246 is shown in spaced relation tocontrol film 244, but in operation would be located on control film 244.The FIG. 33B embodiment differs from that of FIG. 33A in that the sleevereaction chamber 240′ is constructed of a single member and providedwith a circular slot 243′, whereby the tube connector 245′ iscylindrical. Also, in the FIG. 33B embodiment, a microfabricatedactuator 247 is located intermediate the reaction chamber 240′ and anenergy coupler 246. By way of example, the energy coupler in FIGS. 32A,33A and 33B may be an electrode contact, radio frequency source, or anelectrical contact to a DC energy source. Thusly, the individualreaction chambers 240 can be strung together as a series of differentfunctionalities. For example, the series of modules could be a heatedreaction chamber, followed by a magnetically actuated chamber, followedby a shape-memory or electrostatic pumping module. In this way theenergy coupler could also be a way to connect to a series of energysources. In the above example that could be an electrical contact to aDC source for resistive heating; a magnetic coil for magnetic actuation,and then an RF source to actuate the pumping mechanism.

FIG. 34 illustrates the integration of a reaction element in amonolithic flow-through system. In this embodiment the micromachinedelement is placed within or adjacent to the block of material in whichthe flow channels are fabricated. As shown, a block of material 250,constructed for example of silicon, metal or polymer is provided with apair of interconnected openings or passageways 251 and 252 with anembedded control element 253, such as a resistive thin-film heater; amagnetic film or coil; or a actuator such as an electrostatic pump,valve, or mixer, located at the intersection of passageways 251 and 252.A pair of control elements 254 and 255, are embedded in an outer surfaceof block 250 and operatively connected to embedded control element 253,with control leads 256 and 257 extending from block 250, for connectionto control sources, such as a resistive thin-film heater; a magneticfilm or coil; or a actuator such as an electrostatic pump, valve, ormixer. A pair of fluid interconnects 258 and 259 are connected topassageways or openings 251 and 252.

It has thus been shown that the present invention provides asilicon-based or non-silicon-based microreaction chamber which can beused in a hand-held instrument or a large high-throughput instrument.The sleeve reaction chamber may be made of various compatible materialsor combinations of these materials. In addition, the invention providesfor insert/liners, test strips, optical detection, and automatic controlfor the microreaction chamber. Thus, the present invention substantiallyadvances the state of the art for PCR and other chemical reactions.

While particular embodiments, materials, parameters, etc. have been setforth to exemplify and explain the principles of the invention, such arenot intended to be limiting. Modifications and changes may becomeapparent to those skilled in the art, and it is intended that theinvention be limited only by the scope of the appended claims.

What is claimed is:
 1. In a microfabricated chemical reactor, theimprovement comprising: a sleeve reaction chamber constructed ofsilicon-based or nonsilicon-based materials; said sleeve reactionchamber including a slot therein for insertion of reaction fluid.
 2. Theimprovement of claim 1, wherein said slot is constructed for insertionof reaction fluid into said sleeve reaction chamber directly, via atube, or via an insert containing the reaction fluid.
 3. The improvementof claim 1, wherein said sleeve reaction chamber materials are selectedfrom the group consisting of silicon, glass, ceramics, metal, metallicalloys, polymers, composites, and combinations thereof.
 4. Theimprovement of claim 3, wherein said chamber material consists ofceramics selected from the group consisting of crystalline,non-crystalline, silicate, and non-silicate based ceramics.
 5. Theimprovement of claim 3, wherein said chamber material consists ofpolymer material comprising at least one polymer containing a dopant toincrease thermal conduction.
 6. The improvement of claim 1, wherein saidsleeve reaction chamber is provided with at least one optical window. 7.The improvement of claim 1, additionally including at least one via,channel, or hole in said reaction chamber for placement of a detectionsystem.
 8. The improvement of claim 7, additionally including at leastone integrated optical lens located in said at least one via, channel,or hole to provide optical access to reaction chamber liners located insaid reaction chamber.
 9. The improvement of claim 1, wherein saidsleeve reaction chamber is composed of a plurality of bonded members.10. The improvement of claim 9, wherein said bonded members areconstructed of polysilicon and bulk silicon.
 11. The improvement ofclaim 3, wherein said sleeve reaction chamber is composed of a firstmaterial, and including a liner in said reaction chamber and constructedof a second material.
 12. The improvement of claim 11, wherein saidsleeve reaction chamber is composed of material selected from the groupconsisting of silicon and metal, and wherein said liner is composed of apolymer passivation layer.
 13. The improvement of claim 12, wherein saidpolymer passivation layer is composed of teflon or polypropylene. 14.The improvement of claim 11, wherein said sleeve reaction chamber iscomposed of a material selected from the group consisting of silicon,metal, and ceramic, and wherein said liner is composed of a materialselected from the group consisting of polymer, glass, and metal.
 15. Theimprovement of claim 1, wherein said sleeve reaction chamber isconstructed of a first material and includes a heating means composed ofa second material.
 16. The improvement of claim 1, additionallyincluding heating and cooling means for said reaction chamber composedof a thermal electric film.
 17. The improvement of claim 1, wherein saidreaction chamber additionally includes electroactive films forelectrical control of electrically active reagents.
 18. The improvementof claim 1, wherein said reaction chamber additionally include films forcontrol of magnetic and paramagnetic reagents.
 19. The improvement ofclaim 15, wherein said sleeve reaction chamber includes a plurality ofwindows and a heating means located adjacent each window.
 20. Theimprovement of claim 15, wherein said reaction chamber is composed ofmaterial selected from the group of silicon, polymer, and ceramic, andsaid heating means is composed of polysilicon or metal.
 21. Theimprovement of claim 1, additionally including an insert adapted to beinserted into said slot, said insert containing reaction fluids.
 22. Theimprovement of claim 1, wherein said sleeve reaction chamber is providedwith a plurality of slots therein, each slot having at least one window.23. The improvement of claim 22, wherein at least one of said windows isprovided with a test strip.
 24. The improvement of claim 22,additionally including an insert adapted to be inserted into at leastone of said slots, said at least one insert including at least onewindow therein.
 25. The improvement of claim 1, wherein said sleevereaction chamber is constructed and is adapted to be inserted into ahand-held, battery-operated instrument.
 26. The improvement of claim 1,wherein said sleeve reaction chamber is constructed and is adapted to beinserted into an instrument constructed to contain an array of suchreaction chambers.
 27. The improvement of claim 26, wherein saidinstrument containing an array of such reaction chambers is operativelyconnected via an array of microinjectors to a microelectrophoresisarray.
 28. The improvement of claim 26, wherein said instrumentcontaining an array of such reaction chambers is connected directly to amicroelectrophoresis array.
 29. The improvement of claim 27, whereinsaid array of such reaction chambers are constructed of silicon, metalsand ceramic, and wherein said microelectrophoresis array is constructedof glass, polymer and ceramic.
 30. The improvement of claim 6,additionally including an optical detector positioned adjacent saidoptical window.
 31. The improvement of claim 30, additionally includinga data readout system operatively connected to said optical detector,and an instrument controller operatively connected to said data readoutsystem and said reaction chamber.
 32. The improvement of claim 27,wherein each of said reaction chambers include a liner composed ofpolymer, glass, or other compatible material.
 33. The improvement ofclaim 1, additionally including multiple detection systems.
 34. Theimprovement of claim 1, additionally including at least one detectionsystem.
 35. The improvement of claim 34, wherein said at least onedetection system is selected from the group consisting of optical,electochemical, electrocheminescence, magnetic and capacitive.
 36. Theimprovement of claim 1, wherein said reaction chamber additionallyincludes microfabricated elements for providing detection of reactionfluid in said sleeve reaction chamber.
 37. The improvement of claim 1,comprising an array of said sleeve reaction chambers interconnected viatubing to an array of reaction chambers, and control elements providinga flow-through reaction system.
 38. The improvement of claim 1,comprising an array of said sleeve reaction chambers containing an arrayof adjacent detection systems, said detection systems including meansfor providing feedback control of a reaction process in said reactionchambers.